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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 鄭景暉 | |
| dc.contributor.author | I-Hua Wu | en |
| dc.contributor.author | 吳怡樺 | zh_TW |
| dc.date.accessioned | 2021-06-13T05:57:45Z | - |
| dc.date.available | 2012-10-05 | |
| dc.date.copyright | 2011-10-05 | |
| dc.date.issued | 2011 | |
| dc.date.submitted | 2011-07-26 | |
| dc.identifier.citation | Abe S, Yamaguchi S, Watanabe A, Hamada K, Amagasa T (2008). Hard tissue
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Rate and predictability of pulp revascularization in therapeutically reimplanted permanent incisors. Endod Dent Traumatol 2:83-89. Komori T, Yagi H, Nomura S, Yamaguchi A, Sasaki K, Deguchi K, Kitamura Y, Yoshiki S, and Kishimoto T (1997). Targeted disruption of Cbfa1 results in a complete lack of bone formation owing to maturational arrest of osteoblasts. Cell 89:755-764. Komori, T. (2008). Regulation of bone development and maintenance by runx2. Frontiers in Bioscience, 13, 898-903. Lee KS, Hong SH & Bae SC (2002). Both the smad and p38 MAPK pathways play a crucial role in runx2 expression following induction by transforming growth factor-beta and bone morphogenetic protein. Oncogene, 21(47), 7156-7163. Lee KS, Kim HJ, Li QL, Chi XZ, Ueta C, Komori T, Wozney JM, Kim EG, Choi JY, Ryoo HM, & Bae SC (2000). Runx2 is a common target of transforming growth factor β1 and bone morphogenetic protein 2, and cooperation between Runx2 and Smad5 induces osteoblast-specific gene expression in the pluripotent mesenchymal precursor cell line C2C12. Molecular and Cellular Biology, Dec. 8783-8792. Liu J, Jin T, Chang S, Ritchie HH, Smith AJ, Clarkson BH (2007). Matrix and TGF-β-related gene expression during human dental pulp stem cell (DPSC) mineralization. In Vitro Cell. Dev. Biol,－Animal 43: 120-128. MacDougall M, Unterbrink A, Carnes D, Rani S, Luan X, Chen S (2001). Utilization of MO6-G3 immortalized odontoblast cells in studies regarding dentinogenesis. Adv Dent Res August, 15:25-29. Nagashima M (1992). The effects of growth factors on DNA synthesis, proteoglycan synthesis and alkaline phosphatase activity in bonvine dental pulp cells. Archs oral Biol: 37; 3; 231-236. Nagashima M, Nagasawa H, Yamada Y, and HariRedd A (1994). 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Multilineage potential of adult human mesenchymal stem cells. Science 284: 143–147. Shiba H, Fujita T, Doi N, Nakamura S, Nakanishi K, Takemoto T, Hino T, Noshiro M, Kawamoto T, Kurihara H, and Kato Y (1998). J. Cell. Physiol. 174:194-205. Shirakawa M, Shiba H, Nakanishi K, Ogawa T, Okamoto H, Nakashima K, Noshiro M, and Kato Y (1994). Transforming growth factor-beta-1 reduces alkaline phosphatase mRNA and activity and stimulates cell proliferation in cultures of human pulp cells. J Dent Res 73(9): 1509-1514, September. Sloan AJ, Couble ML, Bleicher F, Magloire H, Smith AJ and Farges JC (2001). Expression of tgf-beta receptors i and ii in the human dental pulp by in situ hybridization. Adv Dent Res, 15,63-67. Sloan AJ, Rutherford RB, & Smith AJ (2000). Stimulation of the rat dentine-pulp complex by bone morphogenetic protein-7 in vitro. Arch Oral Biol, 45(2), 173-177. Sonoyama W, Liu Y, Yamaza T, Tuan RS, Wang S, Shi S, Huang GT (2006). Mesenchymal stem cell-mediated functional tooth regeneration in swine. PLoS ONE.1:e79. Sonoyama W, Liu Y, Fang D, Yamaza T, Seo BM, Zhang C, Liu H, Gronthos S, Wang CY, Wang S, Shi S (2008). Characterization of the apical papilla and its residing stem cells from human immature permanent teeth: a pilot study. J Endod 34:166-171. Sporn MB and Roberts AB (1990). Tgf-beta: Problems and prospects. Cell Regulation 1(12), 875-882. Tziafas D, Kodonas K (2010). Differentiation potential of dental papilla, dental pulp, and apical papilla progenitor cells. J Endod 36:781-9. Unterbrink A, O’Sullivan M, Chen S and MacDougall M (2001). TGF-β1 downregulates DMP-1 and DSPP in odontoblasts. Connective Tissue Research, 43: 354-358. Wu RS and Bonner WH(1981). Separation of basal histone synthesis from S-phase histone synthesis in dividing cells. Cell 27(2 Pt 1): 321-330. Yang ZH, Zang XJ, Dang NN, Ma ZF et al (2009). Apical tooth germ cell-conditioned medium enhances the differentiation of periodontal ligament stem cells into cementum/periodontal ligament-like tissues. J Periodont Res 44:199-210. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/34196 | - |
| dc.description.abstract | 實驗目的: 轉型生長因子（Transforming growth factor-beta, TGF-β）對於細胞生長、細胞分化以及牙本質生成扮演著重要角色，但其訊息傳導路徑還未被完全了解，並且對於牙根尖細胞之特性也尚未了解。本實驗之目的在於探討TGF-β1 對牙根尖細胞生長與分化所造成的影響與機制。
實驗方法：使用TGF-β1 對於人類牙根尖細胞做刺激與培養，在一些 實驗另有先加入抑制劑：SB431542 或U0126 做前處理之後，以MTT、 直接計算活細胞數量、鹼性磷酸酶染色及活性測定（ALP staining & activity quantitative assay ）、膠原蛋白定量測定（Sircol collagen assay）、Alizarin Red 染色、鈣離子定量測定以及反轉錄聚合酶反應（RT-PCR）等實驗，來觀察我們的發現。 實驗結果：在細胞生長或是細胞外基質（extracellular matrix）方 面，TGF-β1 主要是藉由Smad 2/3 的路徑，刺激人類牙根尖細胞的細胞存活率以及膠原蛋白的生成；並且，TGF-β1 在較低的濃度下（0.5-1ng/ml）會促進鹼性磷酸酶的活性，在較高濃度（5-10 ng/ml）則是有抑制的效果，並且ERK 的抑制劑U0126 可逆轉低濃度時的促進效果，卻無法逆轉其高濃度的抑制效果，這代表了TGF-β1 在不同的濃度下會引發不同的訊息傳導途徑。而在鈣化誘導方面，TGF-β1 則是有抑制鈣化小節（nodule）以及鈣離子的形成。 結論：TGF-β1 在人類牙根尖細胞中的訊息傳遞是相當複雜的，除了常見的Smad 2/3 途徑之外，還包含了ERK 等其他Non-Smad 途徑。經 由ALP 活性測定實驗我們可得知，TGF-β1 會隨著濃度的不同而對細 胞有著相異的效果，並且濃度不同所引發訊息傳導途徑也會有所不 同。種種的觀察對於人類牙根尖細胞未來是否有機會運用在牙根再 生，有某種程度上的進ㄧ步理解。 | zh_TW |
| dc.description.abstract | Aim：Transforming growth factor β1 (TGF-β1) plays an important role in cell proliferation, matrix formation, and osteogenesis / odontogenesis. The purpose of this
study is to investigate the effects of TGF-β1 on human apical papilla cells. We hypothesize that TGF-β1 can stimulate the two signaling pathways, MEK/ERK and Smad 2/3 to mediate alkaline phosphatase (ALP) expression, collagen matrix deposition and calcium deposition in human apical papilla cells. Materials and Methods：Primary-cultured human apical papilla cells were treated with TGF-β1. In some experiments, apical papilla cells were pretreated with SB431542 ( an ALK5 / Smad 2/3 inhibitor) or U0126 ( a MEK/ERK inhibitor) 30 minutes before adding TGF-β1. Cell viability and cell growth were examined by MTT assay or direct counting of viable cell numbers. Collagen content was determined by Sircol Collagen assay. Cell differentiation and mineralization were evaluated by alkaline phosphatase（ALP）staining, ALP activity quantitative assay, Alizarin Red staining, and calcium quantitative analysis. Changes in mRNA expression were determined by reverse-transcription Polymerase Chain Reaction（RT-PCR）. Results：In human apical papilla cells, TGF-β1 increased cell numbers and cell viability. Cells under the treatment of TGF-β1 (>0.1 ng/ml) would induce collagen formation. Pretreatment of U0126（a MEK/ERK inhibitior）was not effective to reverse the effects of TGF-β1 on cell growth and matrix formation; but SB431542（an ALK5/ Smad 2/3 inhibitor）could prevent those effects. In the differentiation, TGF-β1 down-regulates ALP activity in the lower concentration （0.5-1 ng/ml) and up-regulates it in the higher concentration（5-10 ng/ml）. SB431542 can reverse the effect of TGF-β1 at lower or higer concentration; but U0126 only can reverse the effect of TGF-β1 at lower concentration. Besides, TGF-β1 inhibits the mineralization on apical papilla cells. Conclusion：Signal transduction of TGF-β1 in human apical papilla cells is complex. Different cell sources, different concentrations of growth factor, presence of absence of serum or culture conditions can affect the effects of TGF-β1. Besides, the well-known Smad 2/3 pathway, non Smad pathway（ex. ERK）may also take part in this complex system. These results highlight our future use of growth factors in pulpal repair and dentinogenesis. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-13T05:57:45Z (GMT). No. of bitstreams: 1 ntu-100-R97422025-1.pdf: 2300706 bytes, checksum: 7b6b830cb930eef8fe2bd2a598d13f95 (MD5) Previous issue date: 2011 | en |
| dc.description.tableofcontents | 中文摘要………………………………………………………………1
Abstract………………………………………………………………3 Chapter I Literature Review…………………………………….…5 1.1 Apical papilla cells……………………………………………5 1.2 Transforming Growth Factor-beta………………………………8 1.2.1 Transforming Growth Factor-beta superfamily……………8 1.2.2 The Structure of TGF-β isoforms…………………………8 1.2.3 Receptors of TGF-β………………………………………………………9 1.2.4 Signal transduction n pathways of TGF-β1……………10 1.2.5 TGF-β1 in dental pulp………………………………………11 1.3 Extracellular matrix……………………………………………12 1.4 Regulation of Alkaline Phosphatase (ALP) and its role in cell differtiation….13 Chapter II The Purposes and Hyphothesis of the Study…………... 15 Chapter III Materials and Methods……………………………16 3.1 Culture of human root apical papilla cells………………17 3.2 Surface marker of SCAP…………………………………………17 3.3 Cell growth analyses……………………………………………18 3.3.1 MTT Assay………………………………………………………18 3.3.2 Directly counting viable cells……………………………18 3.4 Collagen content assay…………………………………………19 3.5 Alkaline phosphatase staining………………………………20 3.6 Alkaline phosphatase activity quantitative assay………20 3.7 Differentiation stimulation…………………………………21 3.7.1 Alizarin Red stain……………………………………………21 3.7.2 Calcium quantitative analysis.......................22 3.8 Reverse Transcription Polymerse Chain Reaction (RT-PCR).........................23 3.8.1 Isolation of total RNA………………………………………23 3.8.2 RNA Quantitation………………………………………………24 3.8.3 Reverse Transcription (RT)…………………………………24 3.8.4 Polymerase Chain Reaction (PCR)…………………………25 3.9 Statistical analysis……………………………………………25 Chapter IV Results……………………………………………………26 4.1 Morphological observation on human apical papilla cells…………………….26 4.2 Surface marker of SCAP…………………………………………26 4.3 Effects of TGF-β1 on cell viability of apical papilla cells - MTT assay……...26 4.4 Effects of TGF-β1 on cell viability of apical papilla cells- directly counting cells…………………………………………27 4.5 Effects of TGF-β1 on collagen formation of apical papilla cells and its modulation by SB431542 and U0126…………………………………………27 4.6 Effect of TGF-β1 on ALP activity of apical papilla cells and its modulation by SB431542 and U0126 - ALP stain……………………………………………28 4.7 Effect of TGF-β1 on ALP activity of apical papilla cells - ALP activity quantitative assay………………………28 4.8 Effect of TGF-β1 on osteogenesis of apical papilla cells- Alizarin Red Stain…………………………………………29 4.9 Effect of TGF-β1 on osteogenesis of apical papilla cells - Calcium quantitative analysis…………………………29 Chapter V Disscussion………………………………………………30 5.1 Effect of TGF-β1 on the viability and proliferation of human apical papilla cells……………………………………… 30 5.2 Effect of TGF-β1 on matrix formation of human apical papilla cells………...32 5.3 Effect of TGF-β1 on the differentiation of human apical papilla cells…………………………………………………33 5.4 Effect of TGF-β1 on the mineralization of human apical papilla cells.............34 Chapter VI Conclusion………………………………………………36 References…………………………………………………………… 37 Figure 1：apical papilla……………………………………………43 Figure 2 & 3：Signal Transduction Pathway of TGF-β superfamily…………………..44 Figure 4：Cell surface marker……………………………………45 Figure 5：Cell morphology…………………………………………46 Figure 6：MTT Assay…………………………………………………47 Figure 7：MTT Assay- AP cell 50000cells/well -Serum free DMEM -TGFβ1+inhibitors SB431542 (1, 2.5 uM)-5days -6 well……………………………………………48 Figure 8：MTT Assay- AP cell 50000cells/well -Serum free DMEM -TGFβ1+inhibitors U0126 (1, 10 uM)-5days -6 ell………………………………………………48 Figure 9：Directly counting cell numbers………………………………49 Figure 10：Sircol Collagen Assay.........................50 Figure 11：Sircol Collagen Assay- AP cell 100000cells/well -Serum free DMEM-TGFβ1+inhibitors SB431542 (1, 2.5 uM)-5days -24 well…………………………….51 Figure 12：Sircol Collagen Assay - AP cell 50000cells/well -Serum free DMEM-TGFβ1+inhibitors U0126 (1, 10 uM)-5days -6 well…………………………………..51 Figure 13：ALP stain (5, 10 days)………………………………52 Figure 14：ALP activity quantitative assay (5, 10 days)………………………………53 Figure 15：ALP stain (with/ without inhibitor- SB431542/ U0126)…………………...54 Figure 16：ALP activity quantitative assay- AP cell 100000 cells/well –DMEM with 10%FBS-TGFβ1 – with/without inhibitor (SB431542 2.5 uM) - 10days -24 well…….55 Figure 17：ALP activity quantitative assay- AP cell 100000 cells/well –DMEM with 10%FBS-TGFβ1 – with/without inhibitor (U0126 uM) - 10days -24 well…………….55 Figure 18：Alizarin Red Stain……………………………………56 Figure 19：Calcium quantitative analysis……………………………………………...57 Figure 20：PCR (BAC、BAX、BCl2)…………………………………58 Figure 21：Preliminary study MTT (10% FBS DMEM)………………………………59 Table 1：PCR Primer Sense Sequences, Antisense Sequence, Base Pairs……………60 Table 2：MTT assay for TGF-ß1 ……………………………………61 Table 3：MTT assay for TGF-ß1 with SB431542 1 & 2.5 uM……62 Table 4：MTT assay for TGF-ß1 with U0126 1 & 10 uM…………63 Table 5：Directly counting viable cells for TGF-ß1…………64 Table 6：Protocol for Sircol Collagen Assay standard sample preparation……………65 Table 7：Sircol Assay for TGF-β1 ………………………………66 Table 8：Sircol Assay for TGF-β1 with SB431542 1 & 2.5 uM ………………………67 Table 9：Sircol Assay for TGF-β1 with U0126 1 & 10 uM……………………………67 Table 10：ALP activity quantitative assay for TGF-β1 (5, 10 days)……………………68 Table 11：Calcium quantitative analysis for TGF-β1 (10, 20, 30 days)………………69 | |
| dc.language.iso | en | |
| dc.subject | 牙根尖細胞 | zh_TW |
| dc.subject | 鈣化 | zh_TW |
| dc.subject | 鹼性磷酸酶 | zh_TW |
| dc.subject | 轉型生長因子 | zh_TW |
| dc.subject | apical papilla cells | en |
| dc.subject | mineralization | en |
| dc.subject | alkaline phosphate | en |
| dc.subject | TGF-β1 | en |
| dc.title | TGF-β1 對於牙根尖細胞生長與分化的影響 | zh_TW |
| dc.title | Effect of TGF-β1 on the growth and differentiation of
human apical papilla cells | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 99-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 陳羿貞,張宏博,李勝揚 | |
| dc.subject.keyword | 牙根尖細胞,轉型生長因子,鹼性磷酸酶,鈣化, | zh_TW |
| dc.subject.keyword | apical papilla cells,TGF-β1,alkaline phosphate,mineralization, | en |
| dc.relation.page | 70 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2011-07-26 | |
| dc.contributor.author-college | 牙醫專業學院 | zh_TW |
| dc.contributor.author-dept | 臨床牙醫學研究所 | zh_TW |
| 顯示於系所單位： | 臨床牙醫學研究所 | |
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